Elеna V. Kuzubova, Alexandra I. Radchenko, Andrey А. Manuylov, Ariana M. Korokina, Natalia V. Koroleva, Ivan A. Yakovlev, Arthur A. Isaev, Roman V. Deev, Mikhail V. Korokin

Assessment of physiological parameters in the application of a double adeno-associated virus 9 with a codon-optimized DYSF gene for limb girdle muscular dystrophy type R2

Introduction

Assessment of physiological parameters in the application of a double adeno-associated virus 9 with a codon-optimized dysf gene for limb girdle muscular dystrophy type r2. Assessing gene therapy for limb girdle muscular dystrophy type R2 using a double AAV9-DYSF virus in mouse models. Shows significant improvement in muscle function & safety.

Abstract

Introduction: Gene therapy for Myoshi myopathy is extremely relevant, as it may become the first pathogenetic treatment for dysferlinopathy. The aim of this study was to study the efficacy and safety of the use of a genetic construct, the AAV9-DYSF-DV3’ virus, for the treatment of limb girdle muscular dystrophy LGMD) type R2. Materials and Methods: Mouse models of limb girdle muscular dystrophy type R2 В6.А-Dysfprmd/GeneJ were used to study the effectiveness of AAV9.DYSF drug and the corresponding C57BL/6J controls were used. During the study, muscle activity was determined on the basis of the following tests: “Grip test”, “Holding an animal on a slippery surface of a vertical rod”, “Forced swimming with a load”, and ”Wire hanging”. In the course of acute and subchronic toxicity, hematological and biochemical blood tests of the rats, histological analysis and ”Open field” behavioral testing were performed. Results and Discussion: In this study, for the first time, a comprehensive investigation of the effectiveness of gene therapy using the two-vector system of adeno-associated AAV9-DYSF-DV3’ virus with overlapping DYSF cDNA sequences was conducted in a mouse model of limb girdle muscular dystrophy type 2 R. Conclusion: During the testing of the drug’s effectiveness, it was discovered that drug AAV9.DYSF showed the best effectiveness in mice with the absence of the protein dysferlin in behavioral testing at the maximum dose (5*1012) with a double intramuscular injection. In the “Grip test”, the index in В6.А-Dysfprmd/GeneJ mice increased by 29% (p=0.0026) relative to that in the K-group. In the tests “Forced swimming with a load”, ”Wire hanging”, and ”Holding an animal on a slippery surface of a vertical rod”, the indicators also improved by 80% (p=0.0019), 104.8% (p=0.001) and 20% (p=0.025), respectively, relative to those of the negative control. During acute and subchronic toxicity, the administration of the drug to animals does not cause death or intoxication.

Review

This study presents an important investigation into a gene therapy approach for limb girdle muscular dystrophy type R2 (LGMD R2), a form of dysferlinopathy. The authors evaluated the efficacy and safety of a novel two-vector adeno-associated virus 9 system (AAV9-DYSF-DV3’) designed to deliver a codon-optimized DYSF gene. Addressing a critical need for pathogenic treatments, the research utilized a relevant mouse model (B6.A-Dysfprmd/GeneJ) to assess improvements in muscle function and overall health. This initial comprehensive exploration of the dual-vector system represents a significant step forward in developing therapeutic strategies for this debilitating genetic disorder. The research demonstrates promising efficacy, with the AAV9-DYSF-DV3’ construct showing statistically significant improvements in multiple behavioral tests indicative of muscle strength and endurance. Notably, treated mice exhibited increases in grip strength, wire hanging time, forced swimming capacity, and performance on a slippery rod test, particularly at the maximum dose and through double intramuscular injection. Furthermore, the study included crucial acute and subchronic toxicity assessments, reporting no animal deaths or signs of intoxication based on hematological, biochemical, histological, and behavioral analyses. This favorable safety profile, combined with the observed functional improvements, strengthens the translational potential of this gene therapy candidate. In conclusion, the findings provide compelling preclinical evidence for the therapeutic viability of the AAV9-DYSF-DV3’ gene therapy in LGMD R2. The observed functional recovery without significant toxicity marks a substantial advance in the quest for effective treatments for dysferlinopathies. Future work should aim to further elucidate the molecular mechanisms of dysferlin restoration, including confirmation of protein expression and localization, alongside comprehensive long-term safety and immunogenicity studies to prepare for eventual clinical translation into human trials.

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